Knockdown of miR-22 targets rescues the MK differentiation defect that results from miR-22 loss. (A-B) We used the CRISPRi (CRISPR interference) approach for knockdown of putative miR-22 target genes in K562:miR-22^KO. K562:scramble and K562:miR-22^KO cell lines were transduced with lentivirus encoding KRAB:dCas9-P2A-mCherry. The KRAB-dCas9 fusion protein is a strong transcriptional repressor that can be targeted by gRNAs to specific sites in the genome. A GFP targeting gRNA was used as a control. Putative miR-22 targets were knocked down by CRISPRi in K562:scramble and K562:miR-22^KO cells, and cells were driven toward megakaryocytic differentiation by treatment with PMA. Extent of differentiation is quantified by DNA content/ploidy (n = 3) (A), and median CD61 expression (n = 5) (B). (C-E) Megakaryocytic differentiation of K562:scramble and K562:miR-22^KO cells by treatment with PMA and assayed for transcript and protein expression. (C) qPCR for miR-22 and GFI1 upon PMA-induced megakaryocytic differentiation. sno202 and GUSB were used as housekeeping genes to quantify relative expression, respectively. Expression is shown relative to scramble clone SCR3 (n = 3). (D) Representative immunoblot against GFI1 in K562:miR-22^KO cells upon PMA-induced megakaryocytic differentiation and in vehicle-treated control. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an endogenous loading control. (E) Quantitation of immunoblot against GFI1 in K562:miR-22^KO cells upon PMA-induced megakaryocytic differentiation and in vehicle treated control. GAPDH was used as an endogenous loading control to quantitate relative expression. Quantified blot is included in supplemental Figure 3C (n = 3). (F) The miR-22 seed sequence containing portion of the GFI1 3′-UTR or a nontargeted control in which the seed sequence was replaced with poly-T tract was cloned downstream of the nanoLuciferase (nanoLuc) gene. The nanoLuc expression vectors were transiently cotransfected with a firefly luciferase expression vector into K562:scramble and K562:miR-22^KO cells, and luminescence was quantified after 48 hours using the Nano-Glo Dual Luciferase Reporter Assay System. Quantitation is luminescence of nanoLuc (experimental vector) over luminescence of firefly luciferase (transfection control) (n = 3). (dT)₇, poly-T tract; KO, knockout; SCR, scramble. *P ≤ .05; **P < .01.